A key problem in vaccine development is finding an effective way to induce cellular immunity. DNA vaccines are constituted by a plasmid encoding the target antigen under the transcriptional control of a promoter region active in human cells. They are good inducers of cellular immune responses however they require a high amount of DNA and three administrations to be effective. Lentiviral vectors (IC-LV) are powerful inducers of cell mediated and humoral immune responses, however they carry the intrinsic risk associated with integration into the host genome. Non-integrating lentiviral vectors (ID-LV) have a mutation in the integrase gene, do not integrate into the genome, nor do they replicate, but are maintained as episomal circular DNA in the nucleus (E-DNA) in the absence of integration. We have previously shown that E-DNA is stable and can express functional proteins. Proteins expressed by ID-LV are recognized by the immune system in cell culture. In addition, ID-LV expressing GFP elicited a long-lasting immune response to GFP after a single intramuscular injection in vivo suggesting that a single administration may be sufficient to induce a protective response. As proof-of-concept of the effectiveness of ID-LV, we will compare the responses induced by ID-LV expressing the NP protein of influenza virus with those obtained with conventional DNA vaccines and IC-LV. Use of influenza virus offers several advantages. First, cell-mediated immunity confers protection from multiple strains towards viral challenge. Second, DNA vaccines have been proven to be effective in inducing protective cell-mediated immunity in animal models. In Aim 1 we will compare humoral and cellular immune response, elicited by immunization with ID-LV, IC-LV and DNA vaccine expressing influenza NP. In Aim 2 we will test the ability of ID-LV to protect the animal from sublethal or lethal challenge and address the question of cross-protection. The influenza mouse model provides the first opportunity to test the efficacy of a protein expressed from ID-LV to protect from a viral challenge in vivo and to compare both protection efficacy and immunologic parameters among the non-integrated and integrated vector and a conventional DNA vaccine. These studies will define the general utility of ID-LV as a delivery vector. If successful, this approach would represent a significant step in vaccine development given the safety feature of non integration. PUBLIC HEALTH RELEVANCE: We have previously shown that proteins expressed by integrase-defective lentiviral vectors (ID-LV) are recognized by the immune system in cell culture and can elicit a long-lasting immune response after a single intramuscular injection in vivo. In this proposal, as proof-of-concept of the effectiveness of ID-LV as a vaccine, we will compare the responses induced by ID-LV expressing the NP protein of influenza virus with those obtained with conventional DNA vaccines and integrase-competent-LV. The influenza mouse model provides the first opportunity to test the efficacy of a protein expressed from ID-LV to protect from a viral challenge in vivo and to compare both protection efficacy and immunologic parameters among the ID-LV and IC-LV and a conventional DNA vaccine. These studies will define the utility of ID-LV as a delivery vector. If successful, this approach would represent a significant step in vaccine development given the safety feature of non-integration.